Terpenoids form the largest class of plant secondary metabolites, comprising more than 55000 compounds. These molecules play very important ecological and physiological functions in plant life including attraction of pollinators, seed disseminators and predators of herbivores, repulsion of herbivores and pathogens, photosynthetic pigments, protein management (prenylation and ubiquitination) and growth regulation (gibberellins) (Vandermoten et al., 2009). In spite of such vast diversity in structure and function, all the terpenoids are derived from the common C5 precursors: dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). These two building blocks are condensed together by a group of enzymes known as isoprenyldiphosphate synthases or short-chain prenyltransferases. Addition of one molecule of IPP to DMAPP yields geranyl pyrophosphate (GPP, C10). Sequential addition of two IPP units to DMAPP results in the production of farnesyl pyrophosphate (FPP, C15); whereas, addition of three IPP molecules to DMAPP yields geranylgeranyl pyrophosphate (GGPP, C20) (Vandermoten et al., 2009). These prenyl pyrophosphates formed by isoprenyldiphosphate synthases are the substrates in turn for terpene synthases, which produce the parent carbon skeletons of the terpenes. Being rich in the terpene flavorants, mango forms an appropriate system to study biosynthesis and regulation of monoterpenes. Farnesyl pyrophosphate synthase (FPPS) is one of the central enzymes in the terpenoid biosynthetic pathway in mango. The genes encoding FPPS have been isolated and characterized from several plant genera such as Lupinus (Attucci et al., 1995), Parthenium (Pan et al., 1996), Oryza (Sanmiya et al., 1997), Lycopersicon (Gaffe et al., 2000; Sallaud et al., 2009) Ginkgo (Wang et al., 2004), Centella (Kim et al., 2005), Taxus (Liao et al., 2006), Picea (Schmidt and Gershenzon, 2007), Chimonanthus (Xiang et al., 2010) and Panax (Kim et al., 2010). The most intensive work on FPPS has been carried out with Arabidopsis (Cunillera et al., 1996; Cunillera et al., 1997, 2000; Delourme et al., 1994), (Masferrer et al., 2002) (Closa et al., 2010; Manzano et al., 2006) and Artemisia (Chen et al., 2000) (Han et al., 2006) (Hemmerlin et al., 2003) (Matsushita et al., 1996) (Banyai et al., 2010) (Zhao et al., 2003). These and other studies provide evidence on the importance of FPPS in providing the precursor (FPP) for sesquiterpene volatiles and also for the higher terpenoids such as dolichols, phytoalexins, sterols, ubiquinones, farnesylated proteins and prenylatedheme group of cytochrome a and a3 (Chappell, 1995; Weinstein et al., 1986). FPP also contributes to the biosynthesis of carotenoids, chlorophylls, tocopherols, gibberellins and geranylgeranylated proteins, when it is involved in the formation of GGPP (Szkopinska and Plochocka, 2005). In fruit, which produces the whole range of these compounds, FPPS could well acts as a key regulatory point in terpenoid biosynthesis as well as an important player in controlling cell cycle progression, growth, development and general metabolism (Chappell, 1995; Gaffe et al., 2000; Grunter et al., 1994). Being such an important component of fruit physiology and metabolism, it has been hypothesized that FPPS plays a key role in the variable fruit quality that mango (Mangifera indica cv. Alphonso) exhibits among′ localities (Kulkarni et al., 2012). This hypothesis is based on the fact that such variation is prominently observed in taste, flavor and texture, as well as the color of the fruit, and FPP might affect the development of each of these properties. For example, glycosylated sesquiterpenes contribute to the taste and flavor of fruits, volatile sesquiterpenes to the odor, carotenoids to the color and sterols to the texture (Chappell, 1995; Clark et al., 1987; Seigler, 1998).
Article titled “Farnesyl pyrophosphate synthase from white lupin: molecular cloning, expression, and purification of the expressed protein” by Attucci et. al published in Arch Biochem Biophys. 1995 Aug. 20; 321(2):493-500, discloses the molecular cloning, expression, and purification of Farnesyl pyrophosphate synthase from white lupin. Two full-length cDNA clones (pFPS1 and pFPS2) were isolated and sequenced, and one of them (pFPS2) was expressed in a bacterial system and the enzyme protein encoded by the clone was purified. The deduced amino acid sequence of lupinfarnesyl pyrophosphate synthase pFPS2 shares 90% and 79% identity with those from Lupinusalbus (pFPS1) and Arabidopsis thaliana, respectively, 51% with the yeast enzyme, and 44% identity with those from rat and human.
Article titled “A cDNA Encoding Farnesyl Pyrophosphate Synthase in White Lupin” by Attucciet. al. published in Plant Physiol. (1995) 108: 835-836 discloses a method of isolating DNA encoding farnesyl pyrophosphate synthase from White Lupin. A λZapIIcDNA library was constructed from poly(A)+ RNA extracted from 10-day-old seedling roots of white lupin (Lupinusalbus). A cDNA clone, pFPS1, which contained an insert of 1354 base pairs, was selected and sequenced. The deduced amino acid sequence of the encoded protein has 80% identity with and 90% similarity to that of FPP synthase identified from A. thaliana (GenBank accession No. X75789).
Article titled “Cloning and sequencing of a cDNA encoding farnesyl pyrophosphate synthase from Gossypium arboretum and its expression pattern in the developing seeds of Gossypium hirsutum” by Liu Chang-Jun et. al. published in Acta Botanica Sinica 1998, Volume 40 (8), 703-710, discloses the isolation of a cDNA encoding farnesyl pyrophosphate synthase from Gossypium arboretum. Nucleotide sequencing revealed that it is a full length cDNA of 1.28 kb and the putative amino acid sequence exhibited 80.7%, 78.9% and 71.6% identities with the FPP synthases of Artimisia annua, Arabidopsis thaliana and Zea mays respectively.
U.S. Pat. No. 6,600,094 titled “Recombinant plant expression vector comprising isolated cDNA nucleotide sequence encoding farnesyl pyrophosphate synthase (FPS) derived from seedlings of sunflower (Helianthus annus)” reports a process of amplifying and sequencing the sunflower FPS cDNAs by using a pair of universal FPS oligonucleotides probe and then expressing the FPS cDNAs in bacterial cells, carrying out functional complementation assay in yeast mutant to verify its function and generating a line of transgenic tobacco plants, in order to observe the influence of the overexpression of sunflower Farnesyl Pyrophosphate Synthase (SFPS) in vivo.
Article titled ‘Expression profiling of various genes during the fruit development and ripening of mango’ by Pandit et. al. published in Plant Physiology and Biochemistry, 48 (2010) explores several flavor related genes along with a few associated to the physiology of developing and ripening in ‘Alphonso’ mango. The temporal and spatial regulation of the genes during development and ripening of ‘Alphonso’ mango has been analysed. Genes implicated in terpenoid metabolism include geranyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase.
As seen from the above disclosures, isolation of farnesyl pyrophosphate synthase (FPPS) which plays an important role in the terpenoid biosynthetic pathway and the nucleotide sequence encoding the same, from mango is not known hitherto and there is a long standing need in the prior art for such sequences. Hence the Inventors have attempted in this research to provide artificial sequences which may be used to impart color, flavor and smell as in natural Alphonso mangoes.